1. Field of the Invention
The present invention relates to vectors, host-vector combinations and processes for preparing stable fusion proteins consisting of a carrier protein and a passenger protein, where expression of the fusion proteins leads to exposure of the passenger domains on the surface of bacterial cells, especially Escherichia coli cells. If required, the passenger domains can be released into the medium by proteases, for example by selected host factors such as, for example, OmpT.
2. Description of Related Art
The exposure of recombinant proteins on the surface of bacterial cells is a method with a large number of possible microbiological, molecular biological, immunological or industrial applications. Production of recombinant proteins in this manner makes their properties, for example binding affinities or enzymatic activities (Francisco et al., Bio. Technology 11 (1993) 491–495) available without a further step such as, for example, disruption of the producer cell being necessary. Since only a limited number of factors are naturally expressed on the bacterial surface, there is in addition specific enrichment of the recombinant protein by comparison with cytosolic production. Another considerable advantage is that the same methods used to select the recombinant protein which is sought can also be used to isolate the producer of this protein, a bacterial cell, and thus a clonal producer which can be permanently stored, stably reproduced and grown on a large scale can be obtained.
Various systems have been used to date for the presentation of recombinant proteins on the cell surface, but these without exception are also used naturally for the transport or secretion of bacterial surface proteins (Little et al., TIBTECH 11 (1993), 3–5). Significantly, in these cases the DNA region which naturally codes for the protein to be transported, the passenger, was replaced or supplemented by the coding DNA region of the required recombinant protein, although the coding regions of the protein domains responsible for the transport, the carrier proteins, usually remained unchanged. It is clear from this that systems in which passenger and carrier components are present immediately adjacent or encoded in one gene, so-called one-component systems, have a considerable advantage by comparison with systems having several independent components (Gentschev et al., Behring Inst. Mitt. 95 (1994) 57–66), especially in the production of universally usable vectors which, besides the property of stable replication, one or more selection markers, and the protein domains needed for transport, must also contain an insertion site for the DNA fragment encoding the passenger. The carrier proteins used in many one-component systems used to date have been E. coli outer membrane proteins. These include, inter alia, LamB (Charbit et al., Gene 70 (1988), 181–189), PhoE (Agterberg et al., Gene 59 (1987), 145–150) or OmpA (Franscisco et al., Proc. Natl. Acad. Sci (1992), 2713–2717), whose use entails disadvantages, however. Thus, additional protein sequences can be integrated only in loops exposed on the surface, which on the one hand leads to fixed amino- and carboxyl-terminal ends on the flanking carrier protein sequences, and on the other hand has a limiting effect on the length of the sequences to be introduced. Although the use of peptidoglycan-associated lipoprotein (PAL) as carrier protein leads to transport to the outer membrane, no presentation of native protein sequences on the surface of E. coli is possible therewith (Fuchs et al., Biol. Technology 9 (1991), 1369–1372). Surface expression of relatively large proteins is possible using a fusion of OmpA and Lpp as carrier protein portion, to whose carboxyl end the passenger protein sequences are attached (Franscisco et al., Proc. Natl. Acad. Sci (1992), 2713–2717). A disadvantage which has to be accepted in this case is that the fixing of the N-terminus of the passenger may prevent correct folding or functioning.
Also known are so-called autotransporter-containing proteins, a family of secreted proteins in Gram-negative bacteria. The publication of Jose et al. (Mol. Microbiol. 18 (1995), 377–382) mentions some examples of such autotransporter proteins. These proteins contain a protein domain which enables an N-terminally attached protein domain to be transported through a pore structure formed from ú-pleated sheet structures in the outer membrane of Gram-negative bacteria. The autotransporter-containing proteins are synthesized as so-called polyprotein precursor molecule. The typical structure of such a precursor protein is divided into three. At the N-terminus there is a signal sequence which is responsible for the transport through the inner membrane, taking advantage of the Sec transport apparatus present in the host and being deleted during this. To this is attached the protein domain to be secreted, followed by a C-terminal helper domain which forms a pore in the outer membrane, through which the N-terminally attached protein domain to be secreted is translocated to the surface. Depending on its function to be carried out, the latter remains there linked to the helper, which is now serving as membrane anchor, on the bacterial surface, or is deleted by proteolytic activity, and this proteolytic activity may be intrinsic to the protein domain to be secreted or be a property derived from the host or be an external/specifically added activity (for example thrombin, IgA protease). Secretion of heterologous polypeptides or proteins using an expression system based on an autotransporter is known. Thus, for example, it is known from EP-A-0 254 090 or the publication of Klauser et al. (EMBO J. 11 (1992), 2327–2335) that the helper domain of the IgA protease from N. gonorrhoeae can express heterologous poly-peptides as passenger domains in the heterologous bacterial strains E. coli and Salmonella typhimurium. 
In addition, the extracellular transport of the protein VirG by shigella is described in Suzuki et al. (J. Biol. Chem. 170 (1995) 30874–30880). This protein is likewise an IgA protease-like autotransporter which is capable of the expression of foreign polypeptides such as, for example, MalE and PhoA, which have been covalently linked to the N terminus of the auto-transporter domain of VirG. In addition, the paper by Shimada et al. (J. Biochem, 116 (1994), 327–334) describes the extracellular transport of a heterologous polypeptide, namely pseudoazurin from A. faecales, in E. coli using the autotransporter domain of the serine protease from S. marcescens. 
In the processes described in the prior art for preparing for the expression of heterologous passenger proteins with the aid of autotransporter systems, however, considerable disadvantages have been found. Thus, on use of the transporter or helper domain of the IgA protease from N. gonorrhoeae in E. coli as host strain, considerable compatibility problems frequently arise. Excessive expression leads to cytolysis or the bacteria show reduced growth even with moderate expression, which in both cases leads to a considerable reduction in the yield of fusion protein and points to weaknesses in the stability of the system. The present invention was thus based on the technical problem of providing carrier proteins which, especially on use of E. coli as host strain, do not lead to these disadvantages because, for a variety of reasons, E. coli is to be preferred to, for example, Neisseria gonorrhoeae as host strain. On the one hand, E. coli strains with recombinant DNA can be cultured even in simple laboratories of safety level 1. In addition, E. coli strains have already been used in the commercial production of recombinant proteins. This means that there is a considerable advantage in the handling and manipulation of recombinant E. coli strains by comparison with other host strains. In addition, a large number of accurately characterized mutant strains of E. coli already exist and permit a selection of the host strain depending on the required use.
This problem is solved by a method for presenting peptides or/and polypeptides on the surface of Gram-negative host bacteria, where    a) there is provision of a host bacterium which is transformed with a vector on which is located, operatively linked to a promoter, a fused nucleic acid sequence comprising:            (i) a signal peptide-encoding nucleic acid section,        (ii) a nucleic acid section coding for the passenger peptide or/and passenger poly-peptide to be presented,        (iii) where appropriate a nucleic acid section coding for a protease recognition site,        (iv) a nucleic acid section coding for a transmembrane linker and        (v) a nucleic acid section coding for a transporter domain of an autotransporter; and            (b) the host bacterium is cultivated under conditions with which there is expression of the fused nucleic acid sequence and presentation of the peptide or polypeptide encoded by the nucleic acid section (ii) on the surface of the host bacterium, characterized in that the nucleic acid section (ii) is heterologous relative to the nucleic acid section coding for the transporter domain (v), and the host bacterium is homologous relative to the nucleic acid section coding for the transporter domain (v).